New human cathepsin

ABSTRACT

The invention provides a new human cathepsin (LCAP) and polynucleotides which identify and encode LCAP. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of LCAP.

FIELD OF THE INVENTION

[0001] This invention relates to nucleic acid and amino acid sequencesof a new human cathepsin and to the use of these sequences in thediagnosis, prevention, and treatment of disorders associated with cellproliferation.

BACKGROUND OF THE INVENTION

[0002] Cathepsins are a family of lysosomal proteases which include thecysteine protease cathepsins B, H, K, L, O2, and S. These enzymes have arole in processes that involve proteolysis and turnover of specificproteins and tissues in local microenvironments. Cathepsins alsoinitiate proteolytic cascades by proenzyme activation, participate inthe expression of functional MHC class II molecules which bind antigenicpeptides, and process antigen in antigen-presenting cells. The variousmembers of this family are differentially expressed, and cathepsin L isclosely associated with monocytes, macrophages, and other cells of theimmune system. The secreted forms of several members of this familyfunction in tissue remodeling through degradation of collagen, laminin,elastin, and other structural proteins and are implicated ininflammation associated with immunological response and in metastasis(Huisman, W. et al. (1974) Biochem. Biophys. Acta 370:297-307; Mizuochi,T. (1994) Immunol. Lett. 43:189-193; and Baldwin, E. T. (1993) Proc.Natl. Acad. Sci. 90:6796-6800).

[0003] The various cathepsin proteases differ in their gene structuresand in their transcriptional regulation. The cathepsin L gene promoterhas no TATA box but includes several SP-1 sites, two AP-2 transcriptionregulatory element binding sites, and a cAMP response element.Experimental data confirm that expression of cathepsin L is induced bymalignant transformation, growth factors, tumor promoters, and cyclicAMP (Troen, B. et al. (1991) Cell Growth Differ. 2:23-31).

[0004] Abnormal regulation and expression of cathepsins is evident invarious inflammatory disease states. In cells isolated from inflamedsynovia, the mRNA for stromelysin, cytokines, TIMP-1, cathepsin,gelatinase, and other molecules is preferentially expressed. Expressionof cathepsins L and D is elevated in synovial tissues from patients withrheumatoid arthritis and osteoarthritis. Cathepsin L expression may alsocontribute to the influx of mononuclear cells which exacerbates thedestruction of the rheumatoid synovium (Keyszer, G. M. (1995) ArthritisRheum. 38:976-984).

[0005] The cathepsins are implicated in several other immune responses.In a rat model of human glomerular disease, the administration of aspecific, irreversible inhibitor of cysteine protease(trans-epoxysuccinyl-L-leucylamido-(3-methyl)butane) significantlyreduced proteinuria (Baricos, W. H. (1991) Arch. Biochem. Biophys.288:468-72). The platelet aggregating cysteine protease implicated inthrombotic thrombocytopenic purpura shows the characteristics of alysosomal cathepsin (Consonni, R. (1994) Br. J. Hematol. 87:321-324). Inaddition, the increased expression and differential regulation of thecathepsins is linked to the metastatic potential of a variety of cancersand as such is of therapeutic and prognostic interest (Chambers, A. F.et al. (1993) Crit. Rev. Oncog. 4:95-114).

[0006] The discovery of a new human cathepsin and the polynucleotidesencoding it satisfies a need in the art by providing compositions whichare useful in the diagnosis, prevention and treatment of disordersassociated with cell proliferation.

SUMMARY OF THE INVENTION

[0007] The invention features a substantially purified polypeptide, anew human cathepsin (LCAP), having the amino acid sequence shown in SEQID NO:1, or fragments thereof.

[0008] The invention further provides an isolated and substantiallypurified polynucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or fragments thereof and acomposition comprising said polynucleotide sequence. The invention alsoprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence encoding the amino acidsequence SEQ ID NO:1, or fragments of said polynucleotide sequence. Theinvention further provides a polynucleotide sequence comprising thecomplement of the polynucleotide sequence encoding the amino acidsequence of SEQ ID NO:1, or fragments or variants of said polynucleotidesequence.

[0009] The invention also provides an isolated and purified sequencecomprising SEQ ID NO.2 or variants thereof. In addition, the inventionprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence of SEQ ID NO:2.

[0010] In another aspect the invention provides a composition comprisingan isolated and purified polynucleotide sequence comprising thecomplement of SEQ ID NO:2, or fragments or variants thereof. Theinvention also provides a polynucleotide sequence comprising thecomplement of SEQ ID NO:2.

[0011] The present invention further provides an expression vectorcontaining at least a fragment of any of the claimed polynucleotidesequences. In yet another aspect, the expression vector containing thepolynucleotide sequence is contained within a host cell.

[0012] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding LCAP under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

[0013] The invention also provides a pharmaceutical compositioncomprising a substantially purified LCAP having the amino acid sequenceof SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.

[0014] The invention also provides a purified antagonist which decreasesthe activity of a polypeptide of SEQ ID NO:1. In one aspect theinvention provides a purified antibody which binds to a polypeptidecomprising at least a fragment of the amino acid sequence of SEQ IDNO:1.

[0015] Still further, the invention provides a purified agonist whichmodulates the activity of the polypeptide of SEQ ID NO:1.

[0016] The invention also provides a method for treating or preventing acancer comprising administering to a subject in need of such treatmentan effective amount of an antagonist which decreases the activity ofLCAP.

[0017] The invention also provides a method for treating or preventingan immune response comprising administering to a subject in need of suchtreatment an effective amount of an antagonist which decreases theactivity of LCAP.

[0018] The invention also provides a method for detecting apolynucleotide which encodes LCAP in a biological sample comprising thesteps of: a) hybridizing a polynucleotide sequence complementary to thepolynucleotide encoding SEQ ID NO:1 to nucleic acid material of abiological sample, thereby forming a hybridization complex; and b)detecting the hybridization complex, wherein the presence of the complexcorrelates with the presence of a polynucleotide encoding LCAP in thebiological sample. In a preferred embodiment, prior to hybridization,the nucleic acid material of the biological sample is amplified by thepolymerase chain reaction.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIGS. 1A, 1B, 1C and 1D show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of a new human cathepsin. Thealignment was produced using MacDNASIS PRO™ software (Hitachi SoftwareEngineering Co. Ltd. San Bruno, Calif.).

[0020]FIGS. 2A and 2B show the amino acid sequence alignments among LCAP(SEQ ID NO:1), rat lysophospholipase (GI 1552244; SEQ ID NO:3) and P.fluorescens carboxyesterase (GI 244501, SEQ ID NO:4) produced using themultisequence alignment program of DNASTAR™ software (DNASTAR Inc,Madison Wis.).

DESCRIPTION OF THE INVENTION

[0021] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0022] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0023] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0024] Definitions

[0025] LCAP, as used herein, refers to the amino acid sequences ofsubstantially purified LCAP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0026] The term “agonist”, as used herein, refers to a molecule which,when bound to LCAP, increases or prolongs the duration of the effect ofLCAP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of LCAP.

[0027] An “allele” or “allelic sequence”, as used herein, is analternative form of the gene encoding LCAP. Alleles may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given natural or recombinant gene may have none, one, ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0028] “Altered” nucleic acid sequences encoding LCAP as used hereininclude those with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent LCAP. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding LCAP,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding LCAP. The encoded protein may also be “altered” and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent LCAP.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of LCAP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

[0029] “Amino acid sequence” as used herein refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of LCAP arepreferably about 5 to about 15 amino acids in length and retain thebiological activity or the immunological activity of LCAP. Where “aminoacid sequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

[0030] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer. aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0031] The term “antagonist” as used herein, refers to a molecule which,when bound to LCAP, decreases the amount or the duration of the effectof the biological or immunological activity of LCAP. Antagonists mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich decrease the effect of LCAP.

[0032] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind LCAPpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

[0033] The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0034] The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

[0035] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic LCAP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0036] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

[0037] A “composition comprising a given polynucleotide sequence” asused herein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding LCAP (SEQ ID NO:1) or fragments thereof (e.g., SEQ ID NO:2 andfragments thereof) may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

[0038] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, has been extendedusing XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′direction and resequenced, or has been assembled from the overlappingsequences of more than one Incyte Clone using a computer program forfragment assembly (e.g., GELVIEW™ Fragment Assembly system, GCG,Madison, Wis.). Some sequences have been both extended and assembled toproduce the consensus sequence.

[0039] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of mRNA encoding LCAP in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein.

[0040] A “deletion”, as used herein, refers to a change in the aminoacid or nucleotide sequence and results in the absence of one or moreamino acid residues or nucleotides.

[0041] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to LCAP or theencoded LCAP. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

[0042] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

[0043] Human artificial chromosomes (HACs) are linear microchromosomeswhich may contain DNA sequences of 10K to 10M in size and contain all ofthe elements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

[0044] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0045] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0046] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0047] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

[0048] “Microarray” refers to an array of distinct polynucleotides oroligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

[0049] The term “modulate”, as used herein, refers to a change in theactivity of LCAP. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional or immunological properties of LCAP.

[0050] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments thereof,and to DNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand.“Fragments” are those nucleic acid sequences which are greater than 60nucleotides than in length, and most preferably includes fragments thatare at least 100 nucleotides or at least 1000 nucleotides, and at least10,000 nucleotides in length.

[0051] The term “oligonucleotide” refers to a nucleic acid sequence ofat least about 6 nucleotides to about 60 nucleotides, preferably about15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides,which can be used in PCR amplification or hybridization assays. As usedherein, oligonucleotide is substantially equivalent to the terms“amplimers”,“primers”, “oligomers”, and “probes”, as commonly defined inthe art.

[0052] “Peptide nucleic acid”, PNA as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least five nucleotides in length linked to a peptide backbone ofamino acid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

[0053] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from five amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length LCAP and fragments thereof.

[0054] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding LCAP,or fragments thereof, or LCAP itself may comprise a bodily fluid,extract from a cell, chromosome, organelle, or membrane isolated from acell, a cell, genomic DNA, RNA, or cDNA(in solution or bound to a solidsupport, a tissue, a tissue print, and the like.

[0055] The terms “specific binding” or “specifically binding”, as usedherein, refers to that interaction between a protein or peptide and anagonist, an antibody and an antagonist. The interaction is dependentupon the presence of a particular structure (i.e., the antigenicdeterminant or epitope) of the protein recognized by the bindingmolecule. For example, if an antibody is specific for epitope “A”, thepresence of a protein containing epitope A (or free, unlabeled A) in areaction containing labeled “A” and the antibody will reduce the amountof labeled A bound to the antibody.

[0056] The terms “stringent conditions” or “stringency”, as used herein,refer to the conditions for hybridization as defined by the nucleicacid, salt, and temperature. These conditions are well known in the artand may be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

[0057] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0058] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0059] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0060] A “variant” of LCAP, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Analogousminor variations may also include amino acid deletions or insertions, orboth. Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software.

[0061] The Invention

[0062] The invention is based on the discovery of a new human cathepsin(hereinafter referred to as “LCAP”), the polynucleotides encoding LCAP,and the use of these compositions for the diagnosis, prevention, ortreatment of disorders associated with cell proliferation.

[0063] Nucleic acids encoding the LCAP of the present invention werefirst identified in Incyte Clone 347021 from a thymus cDNA library(THYMNOT02) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones 347021and 389479 from THYMNOT02 and 2554720 and 2555589 from THYMNOT03.

[0064] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIG. 1.LCAP is 335 amino acids in length and is the preproform of the enzyme.LCAP has three potential N glycosylation sites at N2, N221 and N292,five potential phosphorylation sites at T35, T84, T155, S160, and S271,and three potential thiol protease active sites at Q132 KQCGSCWAFSA,L275DHGVLVVGYG, and Y296WLVKNSWGPEWGSNGYVK. The catalytic residue foreach of the thiol protease active sites is underlined. As shown in FIG.2, LCAP has chemical and structural homology with human and pigcathepsins, GI 29715, SEQ ID NO:3 and GI 1468964, SEQ ID NO:4,respectively. In particular, LCAP shares 77% identity with humancathepsin, and 81% identity with pig cathepsin. Northern analysis showsthe expression of this sequence in 211 cDNA libraries, 75% of which areassociated with cell proliferation; in particular, 37%, with cancer;25%, with immune response; and 13%, with fetal or infant development.

[0065] The invention also encompasses LCAP variants which retain atleast one biological, structural or other functional characteristic ofLCAP. A preferred LCAP variant is one having at least 80%, and morepreferably 90%, amino acid sequence identity to the LCAP amino acidsequence (SEQ ID NO:1). A most preferred LCAP variant is one having atleast 95% amino acid sequence identity to SEQ ID NO:1.

[0066] The invention also encompasses polynucleotides which encode LCAP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of LCAP can be used to produce recombinant molecules whichexpress LCAP. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIG. 1.

[0067] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding LCAP, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring LCAP, and all such variations are to beconsidered as being specifically disclosed.

[0068] Although nucleotide sequences which encode LCAP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring LCAP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding LCAP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding LCAP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0069] The invention also encompasses production of DNA sequences, orfragments thereof, which encode LCAP and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding LCAP or any fragment thereof.

[0070] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency as taught in Wahl, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

[0071] Methods for DNA sequencing which are well known and generallyavailable in the art and may be used to practice any of the embodimentsof the invention. The methods may employ such enzymes as the Klenowfragment of DNA polymerase I Sequenase® (US Biochemical Corp, Cleveland,Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase(Amersham, Chicago, Ill.), or combinations of polymerases andproofreading exonucleases such as those found in the ELONGASEAmplification System marketed by Gibco/BRL (Gaithersburg, Md.).Preferably, the process is automated with machines such as the HamiltonMicro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200;MJ Research, Watertown, Mass.) and the ABI Catalyst and 373 and 377 DNASequencers (Perkin Elmer).

[0072] The nucleic acid sequences encoding LCAP may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0073] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

[0074] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

[0075] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0076] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

[0077] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled devise camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. Genotyper™ andSequence Navigator™, Perkin Elmer) and the entire process from loadingof samples to computer analysis and electronic data display may becomputer controlled. Capillary electrophoresis is especially preferablefor the sequencing of small pieces of DNA which might be present inlimited amounts in a particular sample.

[0078] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode LCAP may be used in recombinant DNAmolecules to direct expression of LCAP, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressLCAP.

[0079] As will be understood by those of skill in the art, it may beadvantageous to produce ABBR-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0080] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterLCAP encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0081] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding LCAP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of LCAP activity, it may be useful toencode a chimeric LCAP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the LCAP encoding sequence and theheterologous protein sequence, so that LCAP may be cleaved and purifiedaway from the heterologous moiety.

[0082] In another embodiment, sequences encoding LCAP may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of LCAP, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

[0083] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of LCAP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0084] In order to express a biologically active LCAP, the nucleotidesequences encoding LCAP or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0085] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding LCAPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0086] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding LCAP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0087] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene,LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding LCAP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0088] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for LCAP. For example, whenlarge quantities of LCAP are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding LCAP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0089] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0090] In cases where plant expression vectors are used, the expressionof sequences encoding LCAP may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruizi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

[0091] An insect system may also be used to express LCAP. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding LCAPmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of LCAP will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which LCAP may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0092] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding LCAP may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing LCAP in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0093] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

[0094] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding LCAP. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding LCAP, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0095] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0096] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress LCAP may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0097] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, βglucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0098] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding LCAP isinserted within a marker gene sequence, transformed cells containingsequences encoding LCAP can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding LCAP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0099] Alternatively, host cells which contain the nucleic acid sequenceencoding LCAP and express LCAP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0100] The presence of polynucleotide sequences encoding LCAP can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding LCAP.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding LCAP todetect transformants containing DNA or RNA encoding LCAP.

[0101] A variety of protocols for detecting and measuring the expressionof LCAP, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson LCAP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0102] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding LCAPinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding LCAP, or any fragments thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland,Ohio). Suitable reporter molecules or labels, which may be used for easeof detection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0103] Host cells transformed with nucleotide sequences encoding LCAPmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode LCAP may be designed to contain signal sequences which directsecretion of LCAP through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join sequences encoding LCAP tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and LCAP may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingLCAP and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying LCAP from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0104] In addition to recombinant production, fragments of LCAP may beproduced by direct peptide synthesis using solid-phase techniquesMerrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of LCAP may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0105] Therapeutics

[0106] Chemical and structural homology exits among LCAP (SEQ ID NO:1)human and pig cathepsins (SEQ ID NO:3 and SEQ ID NO:4, respectively).Expression of LCAP appears to be associated with cell proliferation,particularly in cDNA libraries associated with cancer, immune disorders,and fetal/infant development.

[0107] In one embodiment, an antagonist of LCAP, or a fragment or aderivative thereof, may be administered to a subject to prevent or treata disorder associated with cell proliferation. Disorders of cellproliferation include various types of cancer including, but not limitedto, adenocarcinoma, sarcoma, lymphoma, leukemia, melanoma, myeloma,teratocarcinoma, and in particular, cancers of the adrenal gland,bladder, bone, brain, breast, gastrointestinal tract, heart, kidney,liver, lung, ovary, pancreas, paraganglia, parathyroid, prostate,salivary glands, skin, spleen, testis, thyroid, and uterus. In oneaspect, an antibody specific for LCAP may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express LCAP.

[0108] In another embodiment, a vector expressing the complement of thepolynucleotide encoding LCAP, or a fragment or a derivative thereof, maybe administered to a subject to prevent or treat a cancer including, butnot limited to, those described above.

[0109] In another embodiment, an antagonist of LCAP or a fragment or aderivative thereof, may be administered to a subject to prevent or treatan immune disorder. Such disorders include, but are not limited to,Addison's disease, AIDS, adult respiratory distress syndrome, allergies,anemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn'sdisease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polycystic kidney disease, polymyositis, rheumatoid arthritis,scleroderma, Sjögren's syndrome, autoimmune thyroiditis. In one aspect,an antibody specific for LCAP may be used directly as an antagonist, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express LCAP.

[0110] In another embodiment, a vector expressing the complement of thepolynucleotide encoding LCAP, or a fragment or a derivative thereof, maybe administered to a subject to prevent or treat an immune disorderincluding, but not limited to, those described above.

[0111] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0112] Antagonists or inhibitors of LCAP may be produced using methodswhich are generally known in the art. In particular, purified LCAP maybe used to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind LCAP.

[0113] Antibodies to LCAP may be generated using methods that are wellknown in the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0114] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith LCAP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0115] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to LCAP have an amino acid sequence consistingof at least five amino acids and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of LCAP amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0116] Monoclonal antibodies to LCAP may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0117] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceABBR-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0118] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0119] Antibody fragments which contain specific binding sites for LCAPmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0120] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between LCAP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering LCAP epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0121] In another embodiment of the invention, the polynucleotidesencoding LCAP, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding LCAP may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding LCAP. Thus, complementary molecules or fragments may be used tomodulate LCAP activity, or to achieve regulation of gene function. Suchtechnology is now well known in the art, and sense or antisenseoligonucleotides or larger fragments, can be designed from variouslocations along the coding or control regions of sequences encodingLCAP.

[0122] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encoding LCAP.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

[0123] Genes encoding LCAP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes LCAP. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0124] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′ or regulatory regions of the geneencoding LCAP (signal sequence, promoters, enhancers, and introns).Oligonucleotides derived from the transcription initiation site, e.g.,between positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0125] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding LCAP.

[0126] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0127] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding LCAP. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA constitutively or inducibly can be introduced into cell lines,cells, or tissues.

[0128] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0129] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo. in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposome injectionsor polycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

[0130] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0131] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of LCAP,antibodies to LCAP, mimetics, agonists, antagonists, or inhibitors ofLCAP. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0132] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0133] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0134] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0135] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0136] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0137] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0138] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers may also be used for delivery.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0139] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0140] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0141] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0142] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of LCAP, such labeling wouldinclude amount, frequency, and method of administration.

[0143] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0144] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0145] A therapeutically effective dose refers to that amount of activeingredient, for example LCAP or fragments thereof, antibodies of LCAP,agonists, antagonists or inhibitors of LCAP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0146] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reaction sensitivitiesand tolerance/response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, or onceevery two weeks depending on half-life and clearance rate of theparticular formulation.

[0147] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0148] Diagnostics

[0149] In another embodiment, antibodies which specifically bind LCAPmay be used for the diagnosis of conditions or diseases characterized byexpression of LCAP, or in assays to monitor patients being treated withLCAP, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for LCAP includemethods which utilize the antibody and a label to detect LCAP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0150] A variety of protocols including ELISA, RIA, and FACS formeasuring LCAP are known in the art and provide a basis for diagnosingaltered or abnormal levels of LCAP expression. Normal or standard valuesfor LCAP expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to LCAP under conditions suitable for complex formation Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric, means. Quantities of LCAPexpressed in subject, control and disease, samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0151] In another embodiment of the invention, the polynucleotidesencoding LCAP may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof LCAP may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression of LCAP,and to monitor regulation of LCAP levels during therapeuticintervention.

[0152] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding LCAP or closely related molecules, may be used to identifynucleic acid sequences which encode LCAP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding LCAP, alleles, or related sequences.

[0153] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the LCAP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring LCAP.

[0154] Means for producing specific hybridization probes for DNAsencoding LCAP include the cloning of nucleic acid sequences encodingLCAP or LCAP derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0155] Polynucleotide sequences encoding LCAP may be used for thediagnosis of conditions or diseases which are associated with expressionof LCAP. Examples include Addison's disease, AIDS, adult respiratorydistress syndrome, allergies, anemia, asthma, atherosclerosis,bronchitis, cholecystitus, Crohn's disease, ulcerative colitis, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, atrophicgastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia,irritable bowel syndrome, lupus erythematosus, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polycystic kidney disease,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome,autoimmune thyroiditis; and cancers such as adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma. These cancersmay include, but are not limited to, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. Diagnostics based onsequences encoding LCAP may be particularly useful in prenatal diagnosisand identification of mutations in the fetus through amniocentesis. Thepolynucleotide sequences encoding LCAP may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dipstick, pin, ELISA assays or microarraysutilizing fluids or tissues from patient biopsies to detect altered LCAPexpression. Such qualitative or quantitative methods are well known inthe art.

[0156] In a particular aspect, the nucleotide sequences encoding LCAPmay be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding LCAP may be labeled by standard methods, and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding LCAP in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0157] In order to provide a basis for the diagnosis of diseaseassociated with expression of LCAP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes LCAP, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0158] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0159] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0160] Additional diagnostic uses for oligonucleotides designed from thesequences encoding LCAP may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5′→3′) and another with antisense (3′←5′),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

[0161] Methods which may also be used to quantitate the expression ofLCAP include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0162] In further embodiments, oligonucleotides derived from any of thepolynucleotide sequences described herein may be used as targets inmicroarrays. The microarrays can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents.

[0163] In one embodiment, the microarray is prepared and used accordingto the methods described in PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference.

[0164] The microarray is preferably composed of a large number ofunique, single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides which are only 7-10nucleotides in length. The microarray may contain oligonucleotides whichcover the known 5′, or 3′, sequence, sequential oligonucleotides whichcover the full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray. The “pairs” will be identical, except for one nucleotidewhich preferably is located in the center of the sequence. The secondoligonucleotide in the pair (mismatched by one) serves as a control. Thenumber of oligonucleotide pairs may range from two to one million.

[0165] In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. The oligomers are synthesized atdesignated areas on a substrate using a light-directed chemical process.The substrate may be paper, nylon or other type of membrane, filter,chip, glass slide or any other suitable solid support.

[0166] In another aspect, the oligomers may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available devices (slot blot or dot blotapparatus), materials (any suitable solid support), and machines(including robotic instruments) and may contain 8, 24, 96, 384, 1536 or6144 oligonucleotides, or any other multiple between two and one millionwhich lends itself to the efficient use of commercially availableinstrumentation.

[0167] In order to conduct sample analysis using the microarrays, theRNA or DNA from a biological sample is made into hybridization probes.The mRNA is isolated, and cDNA is produced and used as a template tomake antisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices.etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences. mutations, variants, or polymorphisms amongsamples.

[0168] In another embodiment of the invention, the nucleic acidsequences which encode LCAP may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome, to aspecific region of a chromosome or to artificial chromosomeconstructions, such as human artificial chromosomes (HACs), yeastartificial chromosomes (YACs), bacterial artificial chromosomes (BACs),bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0169] Fluorescent in situ hybridization (FISH as described in Verma etal. (1988) Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York, N.Y.) may be correlated with other physical chromosomemapping techniques and genetic map data. Examples of genetic map datacan be found in various scientific journals or at Online MendelianInheritance in Man (OMIM). Correlation between the location of the geneencoding LCAP on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

[0170] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0171] In another embodiment of the invention, LCAP, its catalytic orimmunogenic fragments or oligoneptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenLCAP and the agent being tested, may be measured.

[0172] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to LCAP large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with LCAP, or fragments thereof, and washed. Bound LCAP is thendetected by methods well known in the art. Purified LCAP can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0173] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding LCAPspecifically compete with a test compound for binding LCAP. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with LCAP.

[0174] In additional embodiments, the nucleotide sequences which encodeLCAP may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0175] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0176] I Thymnot02 cDNA Library Construction

[0177] The THYMNOT02 cDNA library was constructed using polyA RNAisolated from the thymus tissue removed from a 3-year old Caucasianmale. First strand cDNA synthesis was accomplished using an oligo d(T)primer/linker which also contained an XhoI restriction site. Secondstrand synthesis was performed using a combination of DNA polymerase I,E. coli ligase and RNase H, followed by the addition of an EcoRI adaptorto the blunt ended cDNA. The EcoRI adapted, double-stranded cDNA wasthen digested with XhoI restriction enzyme to obtain sequences whichwere inserted into the UniZap® vector system (Stratagene). The vectorwhich contains the pBluescript™ phagemid (Stratagene) was transformedinto E. coli host cells strain XL1-BlueMRF™ (Stratagene).

[0178] The phagemid forms of individual cDNA clones were obtained by thein vivo excision process. Enzymes from both pBluescript and acotransformed f1 helper phage nicked the DNA, initiated new DNAsynthesis, and created the smaller, single-stranded circular phagemidmolecules which contained the cDNA insert. The phagemid DNA wasreleased, purified, and used to reinfect fresh host cells (SOLR,Stratagene). Presence of the phagemid which carries the gene forβ-lactamase allowed transformed bacteria to grow on medium containingampicillin.

[0179] II Isolation and Sequencing of cDNA Clones

[0180] Plasmid DNA was released from the cells and purified using theMiniprep Kit (Catalogue # 77468; Advanced Genetic TechnologiesCorporation, Gaithersburg Md.). This kit consists of a 96 well blockwith reagents for 960 purifications. The recommended protocol wasemployed except for the following changes: 1) the 96 wells were eachfilled with only 1 ml of sterile Terrific Broth (Catalog # 22711,Gibco/BRL) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) thebacteria were cultured for 24 hours after the wells were inoculated andthen lysed with 60 μl of lysis buffer; 3) a centrifugation stepemploying the Beckman GS-6R @2900 rpm for 5 min was performed before thecontents of the block were added to the primary filter plate; and 4) theoptional step of adding isopropanol to TRIS buffer was not routinelyperformed. After the last step in the protocol, samples were transferredto a Beckman 96-well block for storage.

[0181] Alternative methods of purifying plasmid DNA include the use ofMAGIC MINIPREPS™ DNA Purification System (Catalogue #A7100, Promega,Madison Wis.) or QIAwell™-8 Plasmid, QIAwell PLUS DNA and QIAwell ULTRADNA Purification Systems (QIAGEN® Chatsworth Calif.).

[0182] The cDNAs were sequenced by the method of Sanger F and A RCoulson (1975; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200(Hamilton, Reno Nev.) in combination with four Peltier Thermal Cyclers(PTC200 from MJ Research, Watertown Mass.) and Applied Biosystems 377 or373 DNA Sequencing Systems (Perkin Elmer) and reading frame wasdetermined.

[0183] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0184] The nucleotide sequences of the Sequence Listing or amino acidsequences deduced from them were used as query sequences againstdatabases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases which contain previously identified and annotated sequenceswere searched for regions of homology (similarity) using BLAST, whichstands for Basic Local Alignment Search Tool (Altschul S F (1993) J MolEvol 36:290-300; Altschul, S F et al (1990) J Mol Biol 215:403-10).

[0185] BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal or plant) origin. Otheralgorithms such as the one described in Smith R F and T F Smith (1992Protein Engineering 5:35-51), incorporated herein by reference, can beused when dealing with primary sequence patterns and secondary structuregap penalties. As disclosed in this application, the minimum length ofthe sequences in the Sequence Listing is 49 nucleotides, and the upperlimit of uncalled bases where N is recorded rather than A, C, G, or T is12%.

[0186] The BLAST approach, as detailed in Karlin and Altschul (1993;Proc Nat Acad Sci 90:5873-7) and incorporated herein by reference,searches matches between a query sequence and a database sequence, toevaluate the statistical significance of any matches found, and toreport only those matches which satisfy the user-selected threshold ofsignificance. In this application, threshold is set at 10-25 fornucleotides and 10-14 for peptides.

[0187] Incyte nucleotide sequence were searched against the GenBankdatabases for pri=primate, rod=rodent, and mam=mammalian sequences, anddeduced amino acid sequences from the same clones are searched againstGenBank functional protein databases, mamp=mammalian, vrtp=vertebrateand eukp=eukaryote, for homology.

[0188] IV Northern Analysis

[0189] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0190] Analogous computer techniques using BLAST (Altschul, S. F. (1993)supra; Altschul, et al. (1990) supra) are used to search for identicalor related molecules in nucleotide databases such as GenBank or theLIFESEQ™ database (Incyte Pharmaceuticals). This analysis is much fasterthan multiple, membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or homologous.

[0191] The basis of the search is the product score which is defined as:$\frac{\% {\quad \quad}{sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}{\quad \quad}{score}}{100}$

[0192] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0193] The results of northern analysis are reported as a list oflibraries in which the transcript encoding LCAP occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0194] V Extension of LCAP Encoding Polynucleotides

[0195] The nucleic acid sequence of the Incyte Clone 347021 was used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length. One primer was synthesized to initiateextension in the antisense direction, and the other was synthesized toextend sequence in the sense direction. Primers were used to facilitatethe extension of the known sequence “outward” generating ampliconscontaining new, unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO 4.06(National Biosciences), or another appropriate program, to be about 22to about 30 nucleotides in length, to have a GC content of 50% or more,and to anneal to the target sequence at temperatures of about 68′ toabout 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

[0196] Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0197] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing theenzyme and reaction mix. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR wasperformed using the Peltier Thermal Cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)

[0198] A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQuick™ (QIAGEN Inc., Chatsworth, Calif.), and trimmedof overhangs using Klenow enzyme to facilitate religation and cloning.

[0199] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2× Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2× Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

[0200] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

[0201] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0202] In like manner, the nucleotide sequence of SEQ ID NO:2 is used toobtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

[0203] VI Labeling and Use of Individual Hybridization Probes

[0204] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1,or Pvu II; DuPont NEN®).

[0205] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

[0206] VII Microarrays

[0207] To produce oligonucleotides for a microarray, the nucleotidesequence described herein is examined using a computer algorithm whichstarts at the 3′ end of the nucleotide sequence. The algorithmidentifies oligomers of defined length that are unique to the gene, havea GC content within a range suitable for hybridization, and lackpredicted secondary structure that would interfere with hybridization.The algorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

[0208] In the alternative, a chemical coupling procedure and an ink jetdevice are used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein byreference). In another alternative, a “gridded” array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

[0209] VIII Complementary Polynucleotides

[0210] Sequence complementary to the ABBR-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring LCAP. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of LCAP, SEQ ID NO:1. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the ABBR-encoding transcript.

[0211] IX Expression of LCAP

[0212] Expression of LCAP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express LCAP in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0213] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of LCAP into the bacterial growth media which can be useddirectly in the following assay for activity.

[0214] IX Demonstration of LCAP Activity

[0215] The characterization of protease activity and specificity isbased on the rate of cleavage of specific peptide substrates anddetermination of an inhibitor profile. Rates of cleavage for cathepsin Lare assessed by incubation of the protease with substrates such asZ-Phe-Arg-AMC or Bz-Val-Lys-Lys-Arg-AFC and by measuring the rate ofrelease of the fluorescent or chromogenic leaving groups. Furtherspecificity of the protease can be examined by titrating specificinhibitors into the cleavage assays and examining the change in the rateof proteolysis. Inhibitors for cathepsin L includetrans-epoxysuccinyl-L-leucylamido-(3-methyl)butane,trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, and chymostatin.

[0216] XI Production of LCAP Specific Antibodies

[0217] LCAP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regions,is described by Ausubel et al. (supra), and others.

[0218] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radio iodinated, goat anti-rabbit IgG.

[0219] XII Purification of Naturally Occurring LCAP Using SpecificAntibodies

[0220] Naturally occurring or recombinant LCAP is substantially purifiedby immunoaffinity chromatography using antibodies specific for LCAP. Animmunoaffinity column is constructed by covalently coupling LCAPantibody to an activated chromatographic resin, such as CNBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0221] Media containing LCAP is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of LCAP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/ABBR binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and LCAP is collected.

[0222] XIII Identification of Molecules Which Interact with LCAP

[0223] LCAP or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled LCAP, washed and any wells withlabeled LCAP complex are assayed. Data obtained using differentconcentrations of LCAP are used to calculate values for the number,affinity, and association of LCAP with the candidate molecules.

[0224] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 4 334 amino acids amino acid single linear THYMNOT02 347021 1 Met AsnLeu Ser Leu Val Leu Ala Ala Phe Cys Leu Gly Ile Ala Ser 1 5 10 15 AlaVal Pro Lys Phe Asp Gln Asn Leu Asp Thr Lys Trp Tyr Gln Trp 20 25 30 LysAla Thr His Arg Arg Leu Tyr Gly Ala Asn Glu Glu Gly Trp Arg 35 40 45 ArgAla Val Trp Glu Lys Asn Met Lys Met Ile Glu Leu His Asn Gly 50 55 60 GluTyr Ser Gln Gly Lys Leu Gly Phe Thr Met Ala Met Asn Ala Phe 65 70 75 80Gly Asp Met Thr Asn Glu Glu Phe Arg Gln Met Met Gly Cys Phe Arg 85 90 95Asn Gln Lys Phe Arg Lys Gly Lys Val Phe Arg Glu Pro Leu Phe Leu 100 105110 Asp Leu Pro Lys Ser Val Asp Trp Arg Lys Lys Gly Tyr Val Thr Pro 115120 125 Val Lys Asn Gln Lys Gln Cys Gly Ser Cys Trp Ala Phe Ser Ala Thr130 135 140 Gly Ala Leu Glu Gly Gln Met Phe Arg Lys Thr Gly Lys Leu ValSer 145 150 155 160 Leu Ser Glu Gln Asn Leu Val Asp Cys Ser Arg Pro GlnGly Asn Gln 165 170 175 Gly Cys Asn Gly Gly Phe Met Ala Arg Ala Phe GlnTyr Val Lys Glu 180 185 190 Asn Gly Gly Leu Asp Ser Glu Glu Ser Tyr ProTyr Val Ala Val Asp 195 200 205 Glu Ile Cys Lys Tyr Arg Pro Glu Asn SerVal Ala Asn Asp Thr Gly 210 215 220 Phe Thr Met Val Ala Pro Gly Lys GluLys Ala Leu Met Lys Ala Val 225 230 235 240 Ala Thr Val Gly Pro Ile SerVal Ala Met Asp Ala Gly His Ser Ser 245 250 255 Phe Gln Phe Tyr Lys SerGly Ile Tyr Phe Glu Pro Asp Cys Ser Ser 260 265 270 Lys Asn Leu Asp HisGly Val Leu Val Val Gly Tyr Gly Phe Glu Gly 275 280 285 Ala Asn Ser AsnAsn Ser Lys Tyr Trp Leu Val Lys Asn Ser Trp Gly 290 295 300 Pro Glu TrpGly Ser Asn Gly Tyr Val Lys Ile Ala Lys Asp Lys Asn 305 310 315 320 AsnHis Cys Gly Ile Ala Thr Ala Ala Ser Tyr Pro Asn Val 325 330 1366 basepairs nucleic acid single linear THYMNOT02 347021 2 CTCAGAGGCTTGTTTGCTGA GGGTGCCTGC GCAGCTGCGA CGGCTGCTGG TTTTGAAAC 60 TGAATCTTTCGCTCGTCCTG GCTGCCTTTT GCTTGGGAAT AGCCTCCGCT GTTCCAAAT 120 TTGACCAAAATTTGGATACA AAGTGGTACC AGTGGAAGGC AACACACAGA AGATTATAG 180 GCGCGAATGAAGAAGGATGG AGGAGAGCAG TGTGGGAAAA GAATATGAAA ATGATTGAC 240 TGCACAATGGGGAATACAGC CAAGGGAAAC TTGGCTTCAC AATGGCCATG AATGCTTTG 300 GTGACATGACCAATGAAGAA TTCAGGCAGA TGATGGGTTG CTTTCGAAAC CAGAAATTA 360 GGAAGGGGAAAGTGTTCCGT GAGCCTCTGT TTCTTGATCT TCCCAAATCT GTGGATTGA 420 GAAAGAAAGGCTACGTGACG CCAGTGAAGA ATCAGAAACA GTGTGGTTCT TGTTGGGCT 480 TTAGTGCGACTGGTGCTCTT GAAGGACAGA TGTTCCGGAA AACTGGGAAA CTTGTCTCC 540 TGAGCGAGCAGAATCTGGTG GACTGTTCGC GTCCTCAAGG CAATCAGGGC TGCAATGGG 600 GCTTCATGGCTAGGGCCTTC CAGTATGTCA AGGAGAACGG AGGCCTGGAC TCTGAGGAT 660 CCTATCCATATGTAGCAGTG GATGAAATCT GTAAGTACAG ACCTGAGAAT TCTGTTGCA 720 ATGACACTGGCTTCACAATG GTCGCACCTG GAAAGGAGAA GGCCCTGATG AAAGCAGTG 780 CAACTGTGGGGCCCATCTCC GTTGCTATGG ATGCAGGCCA TTCGTCCTTC CAGTTCTAA 840 AATCAGGCATTTATTTTGAA CCAGACTGCA GCAGCAAAAA CCTGGATCAT GGTGTTCTG 900 TGGTTGGCTACGGCTTTGAA GGAGCAAATT CGAATAACAG CAAGTATTGG CTCGTCAAA 960 ACAGCTGGGGTCCAGAATGG GGCTCGAATG GCTATGTAAA AATAGCCAAA GACAAGACA 1020 ACCACTGTGGAATCGCCACA GCAGCCAGCT ACCCCAATGT GTGAGCTGAT GGATGGTAG 1080 GAGGAAGGACTTAAGGACAG CATGTCTGGG GAAATTTTAT CTTGAAACTG ACCAAACCT 1140 TATTGTGTAAGATAAACCAG TTGAATCATG GAGGATCCAA GTTGAGATTT TAATTCTTG 1200 ACATTTTTACAAGGGTAAAA TGTTACCACT ACTTTAATTA TTGTTATACA CAGCTTTTG 1260 ATATCAAAGACTCATTGCTT AATTCTAAGA CTTTTGAATT TTCATTTTTT AAAAAGAGT 1320 ACAAAACAGTTTGAAATAAA TTTTAATTCG TATATAAAAA AAAAAA 1366 333 amino acids amino acidsingle linear 29715 GenBank 3 Met Asn Pro Thr Leu Ile Leu Ala Ala PheCys Leu Gly Ile Ala Ser 1 5 10 15 Ala Thr Leu Thr Phe Asp His Ser LeuGlu Ala Gln Trp Thr Lys Trp 20 25 30 Lys Ala Met His Asn Arg Leu Tyr GlyMet Asn Glu Glu Gly Trp Arg 35 40 45 Arg Ala Val Trp Glu Lys Asn Met LysMet Ile Glu Leu His Asn Gln 50 55 60 Glu Tyr Arg Glu Gly Lys His Ser PheThr Met Ala Met Asn Ala Phe 65 70 75 80 Gly Asp Met Thr Ser Glu Glu PheArg Gln Val Met Asn Gly Phe Gln 85 90 95 Asn Arg Lys Pro Arg Lys Gly LysVal Phe Gln Glu Pro Leu Phe Tyr 100 105 110 Glu Ala Pro Arg Ser Val AspTrp Arg Glu Lys Gly Tyr Val Thr Pro 115 120 125 Val Lys Asn Gln Gly GlnCys Gly Ser Cys Trp Ala Phe Ser Ala Thr 130 135 140 Gly Ala Leu Glu GlyGln Met Phe Arg Lys Thr Gly Arg Leu Ile Ser 145 150 155 160 Leu Ser GluGln Asn Leu Val Asp Cys Ser Gly Pro Gln Gly Asn Glu 165 170 175 Gly CysAsn Gly Gly Leu Met Asp Tyr Ala Phe Gln Tyr Val Gln Asp 180 185 190 AsnGly Gly Leu Asp Ser Glu Glu Ser Tyr Pro Tyr Glu Ala Thr Glu 195 200 205Glu Ser Cys Lys Tyr Asn Pro Lys Tyr Ser Val Ala Asn Asp Thr Gly 210 215220 Phe Val Asp Ile Pro Lys Gln Glu Lys Ala Leu Met Lys Ala Val Ala 225230 235 240 Thr Val Gly Pro Ile Ser Val Ala Ile Asp Ala Gly His Glu SerPhe 245 250 255 Leu Phe Tyr Lys Glu Gly Ile Tyr Phe Glu Pro Asp Cys SerSer Glu 260 265 270 Asp Met Asp His Gly Val Leu Val Val Gly Tyr Gly PheGlu Ser Thr 275 280 285 Glu Ser Asp Asn Asn Lys Tyr Trp Leu Val Lys AsnSer Trp Gly Glu 290 295 300 Glu Trp Gly Met Gly Gly Tyr Val Lys Met AlaLys Asp Arg Arg Asn 305 310 315 320 His Cys Gly Ile Ala Ser Ala Ala SerTyr Pro Thr Val 325 330 334 amino acids amino acid single linear 1468964GenBank 4 Met Lys Pro Ser Leu Phe Leu Thr Ala Leu Cys Leu Gly Ile AlaSer 1 5 10 15 Ala Ala Pro Lys Leu Asp Gln Asn Leu Asp Ala Asp Trp TyrLys Trp 20 25 30 Lys Ala Thr His Gly Arg Leu Tyr Gly Met Asn Glu Glu GlyTrp Arg 35 40 45 Arg Ala Val Trp Glu Lys Asn Met Lys Met Ile Glu Leu HisAsn Gln 50 55 60 Glu Tyr Ser Gln Gly Lys His Gly Phe Ser Met Ala Met AsnAla Phe 65 70 75 80 Gly Asp Met Thr Asn Glu Glu Phe Arg Gln Val Met AsnGly Phe Gln 85 90 95 Asn Gln Lys His Lys Lys Gly Lys Val Phe His Glu SerLeu Val Leu 100 105 110 Glu Val Pro Lys Ser Val Asp Trp Arg Glu Lys GlyTyr Val Thr Ala 115 120 125 Val Lys Asn Gln Gly Gln Cys Gly Ser Cys TrpAla Phe Ser Ala Thr 130 135 140 Gly Ala Leu Glu Gly Gln Met Phe Arg LysThr Gly Lys Leu Val Ser 145 150 155 160 Leu Ser Glu Gln Asn Leu Val AspCys Ser Arg Pro Gln Gly Asn Gln 165 170 175 Gly Cys Asn Gly Gly Leu MetAsp Asn Ala Phe Gln Tyr Val Lys Asp 180 185 190 Asn Gly Gly Leu Asp ThrGlu Glu Ser Tyr Pro Tyr Leu Gly Arg Glu 195 200 205 Thr Asn Ser Cys ThrTyr Lys Pro Glu Cys Ser Ala Ala Asn Asp Thr 210 215 220 Gly Phe Val AspIle Pro Gln Arg Glu Lys Ala Leu Met Lys Ala Val 225 230 235 240 Ala ThrVal Gly Pro Ile Ser Val Ala Ile Asp Ala Gly His Ser Ser 245 250 255 PheGln Phe Tyr Lys Ser Gly Ile Tyr Tyr Asp Pro Asp Cys Ser Ser 260 265 270Lys Asp Leu Asp His Gly Val Leu Val Val Gly Tyr Gly Phe Glu Gly 275 280285 Thr Asp Ser Asn Ser Ser Lys Phe Trp Ile Val Lys Asn Ser Trp Gly 290295 300 Pro Glu Trp Gly Trp Asn Gly Tyr Val Lys Met Ala Lys Asp Gln Asn305 310 315 320 Asn His Cys Gly Ile Ser Thr Ala Ala Ser Tyr Pro Thr Val325 330

What is claimed is:
 1. A substantially purified human cathepsincomprising the amino acid sequence of SEQ ID NO:1 or fragments thereof.2. An isolated and purified polynucleotide sequence encoding the humancathepsin of claim
 1. 3. A polynucleotide sequence which hybridizes tothe polynucleotide sequence of claim
 2. 4. A composition comprising thepolynucleotide sequence of claim
 2. 5. An isolated and purifiedpolynucleotide sequence comprising SEQ ID NO:2 or variants thereof.
 6. Acomposition comprising the polynucleotide sequence of claim
 5. 7. Apolynucleotide sequence which is complementary to the polynucleotidesequence of claim 2 or variants thereof.
 8. A composition comprising thepolynucleotide sequence of claim
 7. 9. An expression vector containingat least a fragment of the polynucleotide sequence of claim
 2. 10. Ahost cell containing the vector of claim
 9. 11. A method for producing apolypeptide comprising the amino acid sequence of SEQ ID NO:1, or afragment thereof, the method comprising the steps of: a) culturing thehost cell of claim 10 under conditions suitable for the expression ofthe polypeptide; and b) recovering the polypeptide from the host cellculture.
 12. A pharmaceutical composition comprising a substantiallypurified human cathepsin having the amino acid sequence of SEQ ID NO:1in conjunction with a suitable pharmaceutical carrier.
 13. A purifiedantibody which specifically binds to the polypeptide of claim
 1. 14. Apurified agonist of the polypeptide of claim
 1. 15. A purifiedantagonist of the polypeptide of claim
 1. 16. A method for treating animmune disorder comprising administering to a subject in need of suchtreatment an effective amount of the pharmaceutical composition of claim12.
 17. A method for treating a cancer comprising administering to asubject in need of such treatment an effective amount of the antagonistof claim
 15. 18. A method for detecting a polynucleotide which encodeshuman cathepsin in a biological sample comprising the steps of: a)hybridizing the polynucleotide of claim 7 to nucleic acid material of abiological sample, thereby forming a hybridization complex; and b)detecting said hybridization complex, wherein the presence of saidcomplex correlates with the presence of a polynucleotide encoding humancathepsin in said biological sample.